Hepatic ischemia/reperfusion (I/R) injury produces cell death in a variety of clinical settings. A mystery regarding I/R injury concerns changes that occur during ischemia that predispose to injury after reperfusion. In the previous period of support, we developed the hypothesis that ATP depletion during ischemia inhibits the H+-pumping vacuolar ATPase (V-ATPase) in lysosomes/endosomes to collapse their acidic pH gradient, which in turn causes release of lysosomal Fe2+ into the cytosol without lysosomal rupture. Mitochondria take up this Fe2+ via the mitochondrial Ca2+,Fe2+ uniporter (MCFU) to predispose to iron-catalyzed hydroxyl radical (OH??) formation (Fenton chemistry) with consequent activation of c-Jun N-terminal kinase (JNK), onset of the mitochondrial permeability transition (MPT) and cell death after reperfusion. We also hypothesize that sphingosine accumulates during ischemia, which inhibits cytochrome oxidase to promote generation the OH? precursors, H2O2 and superoxide (O2??), from the respiratory chain after reperfusion. Together these events synergize to cause lethal cell injury. Pursuing these hypotheses, in Specific Aim 1 we will determine whether DMT1 inhibitors prevent translocation of iron from lysosomes to mitochondria during ischemia and prevent oxidative stress, MPT onset and cell death after reperfusion. We have crossed phloxed DMT1 mice with Alb- Cre mice to create a hepatocyte specific DMT1 knockout. We expect that hepatocellular DMT1 deficiency will prevent lysosomal iron mobilization during ischemia and improve survival after IR. We will then assess whether DMT1 inhibitors and DMT1 deficiency decrease reperfusion injury in vivo utilizing intravital multiphoton microscopy. We will also clarify the specific individual roles of mitochondrial MCFU-mediated Fe2+ and Ca2+ uptake in cytoprotection. Specific Aim 2 is based on our experiments showing that JNK inhibition and JNK2 deficiency decrease I/R injury. We will perform experiments with iron chelators, MCU inhibitors, JNK inhibition and knockouts and MPT inhibitors to test the hypothesis that JNK activation after IR is downstream of iron- dependent Fenton chemistry and upstream of MPT onset. In further in vivo experiments, we will perform phosphoproteomics to determine changes in the mitochondrial outer membrane phosphoproteome after IR that are sensitive to JNK inhibition/knockout. Specific Aim 3 is based on preliminary studies showing that sphingosine accumulates during ischemia and inhibits mitochondrial cytochrome oxidase, we hypothesize that sphingosine accumulation during ischemia enhances formation of H2O2 and O2?? from the inhibited respiratory chain to fuel iron catalyzed OH??. We further hypothesize that inhibition of mitochondrial sphingosine kinase-2 (SK2) inhibition prevents sphingosine elimination by phosphorylation to sphingosine-1-phosphate. We will test elements of this hypothesis by measuring sphingosine metabolites, reactive oxygen species and mitochondrial function during I/R in relation to SK2 inhibition and SK2 deficiency. Overall, success of this project will lead to new mechanistic insights into I/R injury, novel drug targets and innovative translatable therapeutic strategies.